The present invention relates to in vivo methods for inducing the differentiation of monocytes into functional dendritic antigen presenting cells and, more particularly, to extracorporeal methods for treating and incubating monocytes to induce such differentiation. In particular, the present invention provides methods for making immunotherapeutic compositions comprising apoptotic or inactivated disease effector agents and functional dendritic cells derived from induced monocytes.
The use of dendritic cells in cancer immunotherapy is presently an area of significant clinical inquiry. Dendritic cells are highly effective in presenting antigens to responding T-cells; however, dendritic cells normally constitute less than one percent of blood mononuclear leukocytes. Accordingly, a number of in vitro methods have been developed to expand populations of dendritic cells to augment anti-cancer immunity. By exposing increased numbers of dendritic cells to antigens on tumor or other disease-causing cells, followed by reintroduction of the antigen-loaded dendritic cells to the patient, presentation of these antigens to responding T-cells can be enhanced significantly.
For example, culturing blood mononuclear leukocytes for eight days in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) produces large numbers of dendritic cells. These cells can then be externally loaded with tumor-derived peptide antigens for presentation to T-cells. Alternatively, the dendritic cells can be transduced to produce and present these antigens themselves. Expanding populations of dendritic cells transduced to produce and secrete cytokines which recruit and activate other mononuclear leukocytes, including T-cells, may be an even more effective method of generating anti-tumor immune responses.
Transducing cultivated dendritic cells to produce a particular generic tumor antigen and/or additional cytokines is labor intensive and expensive. More importantly, this procedure likely fails to produce and present those multiple tumor antigens that may be most relevant to the individual""s own cancer. Several approaches have been proposed to overcome this problem. Hybridization of cultivated autologous dendritic cells with tumor cells would produce tetraploid cells capable of processing and presenting multiple unknown tumor antigens. In a second proposed approach, acid elution of Class I and Class II major histocompatability complexes (MHC) from the surface of malignant cells would liberate a broad spectrum of tumor-derived peptides. These liberated peptides could then be externally loaded onto MHC complexes of autologous cultivated dendritic cells.
Conventional photopheresis is a method of vaccinating patients against leukemic lymphocytes, even when the distinctive tumor antigen(s) is not known. In this method, malignant cells are exposed to photo-activated 8-methoxypsoralen (8-MOP) which enhances cell surface display of Class I MHC-associated tumor antigens. After intravenous return of these altered malignant lymphocytes to the original patient, a potent anti-tumor response may be generated in about 25% of the patients, leading to diminution of the malignant cell population and occasionally long-standing remissions. Experimental studies in mice, in which autologous dendritic cells are first grown in tissue culture and then admixed with the 8-MOP-treated tumor cells, appears to increase the efficacy of conventional photopheresis. In this experimental protocol, tumorigenic mouse T-cells are rendered apoptotic by photopheresis using 8-MOP and exposure to ultraviolet (UV) energy. Following this chemical alteration of the malignant leukocytes, autologous cultured dendritic cells are added to the apoptotic T-cells, and the cell mix is incubated overnight with shaking to maximize contact between the T-cells and the dendritic cells. The apoptotic T-cell/dendritic cell mix has proven to be an effective cellular vaccine in test mice challenged with viable tumorigenic 2B4.11 cells.
While the above-described experimental protocol is apparently more efficient and comprehensive than alternative approaches, it requires extensive ex vivo cellular manipulations over a period of several days. Accordingly, an in vivo procedure which could in a single day provide large numbers of functional dendritic cells and expose those cells to apoptotic tumor cells would greatly simplify the means by which the anti-tumor cellular vaccine could be prepared.
The present invention is based on the convergence of two disparate phenomena: treating monocytes in a manner which induces their differentiation into functional dendritic antigen presenting cells, and treating disease effector agents to render them apoptotic or to inactivate them. By incubating these treated populations together for a period of time sufficient to optimize processing and presentation of disease associated antigens distinctive to the disease effector agents by the dendritic cells, prior to returning the dendritic antigen presenting cells to the patient, clinically enhanced immunity to the disease associated antigens is achieved.
As used herein, the term xe2x80x9cdisease effector agentsxe2x80x9d refers to agents that are central to the causation of a disease state in a subject and which express disease-associated antigens. In certain circumstances, these disease effector agents are disease-causing cells which may be circulating in the bloodstream, thereby making them readily accessible to extracorporeal manipulations and treatments. Examples of such disease-causing cells include malignant T-cells, malignant B cells, T-cells and B cells which mediate an autoimmune response, and virally or bacterially infected white blood cells which express on their surface viral or bacterial peptides or proteins. Exemplary disease categories giving rise to disease-causing cells include leukemia, lymphoma, autoimmune disease, graft versus host disease, and tissue rejection. Disease associated antigens which mediate these disease states and which are derived from disease-causing cells include peptides that bind to a MHC Class I site, a MHC Class II site, or to a heat shock protein which is involved in transporting peptides to and from MHC sites (i.e., a chaperone). Disease associated antigens also include viral or bacterial peptides which are expressed on the surface of infected white blood cells, usually in association with an MHC Class I or Class II molecule.
Other disease-causing cells include those isolated from surgically excised specimens from solid tumors, such as lung, colon, brain, kidney or skin cancers. These cells do not ordinarily circulate in the blood in significant quantity, but can be manipulated extracorporeally in analogous fashion to blood leukocytes, after they are brought into suspension or propagated in tissue culture.
In addition to disease-causing cells, disease effector agents falling within the scope of the invention further include microbes such as bacteria, fungi and viruses which express disease-associated antigens. It should be understood that viruses can be engineered to be xe2x80x9cincompletexe2x80x9d, i.e., produce distinguishing disease-causing antigens without being able to function as an actual infectious agent, and that such xe2x80x9cincompletexe2x80x9d viruses fall within the meaning of the term xe2x80x9cdisease effector agentsxe2x80x9d as used herein.
Accordingly, the present invention provides, in one aspect, a method for inducing the differentiation of monocytes contained in an extracorporeal quantity of a subject""s blood into functional dendritic antigen presenting cells. According to the invention, the monocytes are treated by at least one of the following: (1) exposing the monocytes to physical perturbation, (2) irradiating the monocytes in the presence of a photoactivatable agent capable of forming photoadducts with cellular components, and (3) treating the monocytes with a DNA binding agent. Following treatment, the monocytes are incubated for a period of time sufficient to maximize the number of functional dendritic cells.
In the preferred embodiment of this aspect of the invention, the monocytes are separated from the extracorporeal quantity of the subject""s blood by leukapheresis. Following separation, the monocytes are subjected to photopheresis using 8-MOP and exposure to UV energy. The cells are then incubated for a period of from about 6 to about 48 hours, and most preferably from about 12 to about 24 hours.
The invention provides, in a second aspect, a method of enhancing the presentation of disease associated antigens. According to the method, disease effector agents contained in a quantity of a subject""s blood or derived from an exogenous source and capable of expressing at least one disease associated antigen are treated to render the agents apoptotic or to inactivate them. Monocytes contained in a quantity of the subject""s blood are also treated by at least one of the following: (1) exposing the monocytes to physical perturbation, (2) irradiating the monocytes in the presence of a photoactivatable agent capable of forming photoadducts with cellular components, and (3) treating the monocytes with a DNA cross-linking agent. The treated disease effector agents and the treated monocytes are then incubated together for a period of time sufficient to induce differentiation of the monocytes into functional dendritic antigen presenting cells and to optimize processing and presentation of the disease associated antigens by the dendritic cells
In a preferred embodiment of this aspect of the invention, the monocytes and the disease effector agents are separated from the quantity of the patient""s blood by leukapheresis. The monocytes and disease effector agents are then exposed to photo-activated 8-MOP and incubated together for a period of from about 6 to about 48 hours, and preferably for about 12 to about 24 hours. The mix is then reinfused into the subject to elicit an immune response.